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. 1994 May;68(5):2994–2999. doi: 10.1128/jvi.68.5.2994-2999.1994

Hepadnavirus P protein utilizes a tyrosine residue in the TP domain to prime reverse transcription.

M Weber 1, V Bronsema 1, H Bartos 1, A Bosserhoff 1, R Bartenschlager 1, H Schaller 1
PMCID: PMC236789  PMID: 7512155

Abstract

Hepadnavirus DNA minus strands are covalently linked at their 5' terminus to the viral P gene product, which has been taken to indicate that the hepadnaviral polymerase polypeptide itself also functions as a protein primer for initiating reverse transcription of the RNA pregenome. The present study confirms this indication by identifying the nucleotide-linked amino acid in the P protein sequence of the duck hepatitis B virus (DHBV). In a first set of experiments, mutational analysis of three phylogenetically conserved tyrosine residues in the DNA terminal (TP) domain indicated that of these, only tyrosine 96 was essential for both viral DNA synthesis in transfected cells and priming of DNA synthesis in a cell-free system. This assignment was confirmed by direct biochemical analysis: tryptic peptides from the DHBV P protein, 32P labelled at the priming amino acid by the initiating dGTP and additionally labelled internally by [35S]methionine, were isolated and analyzed in parallel to reference peptides synthesized chemically and 33P labelled by a tyrosine kinase. Mobility in high-performance liquid chromatography, as well as the release in stepwise amino acid sequencing of phospholabel and of [35S]methionine, identified the priming amino acid unequivocally as the tyrosine in the sequence 91KLSGLYQMK99, which is located in the center of the TP domain. Conserved sequence motifs surrounding Tyr-96 allow the prediction of the priming tyrosine in other hepadnaviruses. Weak sequence similarity to picornavirus genome-linked polypeptides (VPgs) and similar gene organization suggest a common origin for the mechanisms that use protein priming to initiate synthesis of viral DNA genomes or RNA genomes from an RNA template.

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Selected References

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  1. Aebersold R., Watts J. D., Morrison H. D., Bures E. J. Determination of the site of tyrosine phosphorylation at the low picomole level by automated solid-phase sequence analysis. Anal Biochem. 1991 Nov 15;199(1):51–60. doi: 10.1016/0003-2697(91)90268-x. [DOI] [PubMed] [Google Scholar]
  2. Bartenschlager R., Junker-Niepmann M., Schaller H. The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation. J Virol. 1990 Nov;64(11):5324–5332. doi: 10.1128/jvi.64.11.5324-5332.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartenschlager R., Schaller H. The amino-terminal domain of the hepadnaviral P-gene encodes the terminal protein (genome-linked protein) believed to prime reverse transcription. EMBO J. 1988 Dec 20;7(13):4185–4192. doi: 10.1002/j.1460-2075.1988.tb03315.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  5. Ganem D., Varmus H. E. The molecular biology of the hepatitis B viruses. Annu Rev Biochem. 1987;56:651–693. doi: 10.1146/annurev.bi.56.070187.003251. [DOI] [PubMed] [Google Scholar]
  6. Gerlich W. H., Robinson W. S. Hepatitis B virus contains protein attached to the 5' terminus of its complete DNA strand. Cell. 1980 Oct;21(3):801–809. doi: 10.1016/0092-8674(80)90443-2. [DOI] [PubMed] [Google Scholar]
  7. Hirsch R. C., Lavine J. E., Chang L. J., Varmus H. E., Ganem D. Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as wel as for reverse transcription. Nature. 1990 Apr 5;344(6266):552–555. doi: 10.1038/344552a0. [DOI] [PubMed] [Google Scholar]
  8. Hirsch R. C., Loeb D. D., Pollack J. R., Ganem D. cis-acting sequences required for encapsidation of duck hepatitis B virus pregenomic RNA. J Virol. 1991 Jun;65(6):3309–3316. doi: 10.1128/jvi.65.6.3309-3316.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jobling S. A., Gehrke L. Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequence. Nature. 1987 Feb 12;325(6105):622–625. doi: 10.1038/325622a0. [DOI] [PubMed] [Google Scholar]
  10. Junker-Niepmann M., Bartenschlager R., Schaller H. A short cis-acting sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA. EMBO J. 1990 Oct;9(10):3389–3396. doi: 10.1002/j.1460-2075.1990.tb07540.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Khudyakov YuE, Makhov A. M. Prediction of terminal protein and ribonuclease H domains in the gene P product of hepadnaviruses. FEBS Lett. 1989 Jan 30;243(2):115–118. doi: 10.1016/0014-5793(89)80110-3. [DOI] [PubMed] [Google Scholar]
  12. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Molnar-Kimber K. L., Summers J., Taylor J. M., Mason W. S. Protein covalently bound to minus-strand DNA intermediates of duck hepatitis B virus. J Virol. 1983 Jan;45(1):165–172. doi: 10.1128/jvi.45.1.165-172.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nassal M., Schaller H. Hepatitis B virus replication. Trends Microbiol. 1993 Sep;1(6):221–228. doi: 10.1016/0966-842x(93)90136-f. [DOI] [PubMed] [Google Scholar]
  15. Radziwill G., Tucker W., Schaller H. Mutational analysis of the hepatitis B virus P gene product: domain structure and RNase H activity. J Virol. 1990 Feb;64(2):613–620. doi: 10.1128/jvi.64.2.613-620.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Salas M. Protein-priming of DNA replication. Annu Rev Biochem. 1991;60:39–71. doi: 10.1146/annurev.bi.60.070191.000351. [DOI] [PubMed] [Google Scholar]
  17. Schlicht H. J., Radziwill G., Schaller H. Synthesis and encapsidation of duck hepatitis B virus reverse transcriptase do not require formation of core-polymerase fusion proteins. Cell. 1989 Jan 13;56(1):85–92. doi: 10.1016/0092-8674(89)90986-0. [DOI] [PubMed] [Google Scholar]
  18. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  19. Summers J., Mason W. S. Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell. 1982 Jun;29(2):403–415. doi: 10.1016/0092-8674(82)90157-x. [DOI] [PubMed] [Google Scholar]
  20. Wang G. H., Seeger C. Novel mechanism for reverse transcription in hepatitis B viruses. J Virol. 1993 Nov;67(11):6507–6512. doi: 10.1128/jvi.67.11.6507-6512.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wang G. H., Seeger C. The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis. Cell. 1992 Nov 13;71(4):663–670. doi: 10.1016/0092-8674(92)90599-8. [DOI] [PubMed] [Google Scholar]
  22. Wimmer E., Hellen C. U., Cao X. Genetics of poliovirus. Annu Rev Genet. 1993;27:353–436. doi: 10.1146/annurev.ge.27.120193.002033. [DOI] [PubMed] [Google Scholar]
  23. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]
  24. Zoulim F., Seeger C. Reverse transcription in hepatitis B viruses is primed by a tyrosine residue of the polymerase. J Virol. 1994 Jan;68(1):6–13. doi: 10.1128/jvi.68.1.6-13.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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